U.S. patent application number 13/000279 was filed with the patent office on 2011-08-04 for method and compositions for inhibition of double stranded dna viruses.
Invention is credited to Paul G. Ahlquist, Paul F. Lambert, Shane Pearce, Dohun Pyeon.
Application Number | 20110189258 13/000279 |
Document ID | / |
Family ID | 41466549 |
Filed Date | 2011-08-04 |
United States Patent
Application |
20110189258 |
Kind Code |
A1 |
Pyeon; Dohun ; et
al. |
August 4, 2011 |
METHOD AND COMPOSITIONS FOR INHIBITION OF DOUBLE STRANDED DNA
VIRUSES
Abstract
A method of inhibiting double-stranded DNA virus infection is
disclosed. In one embodiment, the method involves exposing a
papillomavirus to an effective amount of an inhibitor selected from
the group of G1, S, G2, and M cell cycle inhibitors. In another
embodiment, the method involves administering an inhibitor selected
from the group of G1, S, G2, and M cell cycle inhibitors to a
susceptible tissue or cell.
Inventors: |
Pyeon; Dohun; (Greenwood
Village, CO) ; Lambert; Paul F.; (Madison, WI)
; Pearce; Shane; (Eau Claire, WI) ; Ahlquist; Paul
G.; (Madison, WI) |
Family ID: |
41466549 |
Appl. No.: |
13/000279 |
Filed: |
June 29, 2009 |
PCT Filed: |
June 29, 2009 |
PCT NO: |
PCT/US09/49085 |
371 Date: |
April 15, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61133676 |
Jul 1, 2008 |
|
|
|
Current U.S.
Class: |
424/431 ;
514/263.4; 514/27; 514/274; 514/729 |
Current CPC
Class: |
A61K 31/7048 20130101;
A61K 31/52 20130101; A61P 31/20 20180101; C12N 2710/20011 20130101;
A61K 31/505 20130101; G01N 2500/10 20130101; G01N 2333/025
20130101; A61K 31/047 20130101 |
Class at
Publication: |
424/431 ; 514/27;
514/729; 514/274; 514/263.4 |
International
Class: |
A61F 13/20 20060101
A61F013/20; A61K 31/7048 20060101 A61K031/7048; A61K 31/047
20060101 A61K031/047; A61K 31/513 20060101 A61K031/513; A61K 31/52
20060101 A61K031/52; A61P 31/20 20060101 A61P031/20 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with United States government
support awarded by the following agencies: NIH CA22443, Ahlquist
subproject 144-MQ69, and Lambert subproject 144-MQ67. The United
States government has certain rights in this invention.
Claims
1. A method of inhibiting double-stranded DNA virus infection,
comprising the steps of: (a) identifying an individual in danger of
viral infection, and (b) exposing tissues or cells of the
individual that are susceptible to virus infection to an effective
amount of a viral inhibitor, wherein the inhibitor is selected from
the group of G1, S, G2, and M cell cycle inhibitors.
2. The method of claim 1 wherein the virus is a papovavirus.
3. The method of claim 2 wherein the virus is HPV.
4. The method of claim 3 wherein the virus is high risk HPV.
5. The method of claim 4 wherein the virus is HPV16.
6. The method of claim 3 wherein the virus is low risk HPV.
7. The method of claim 1 wherein the inhibitor is selected from the
group consisting of etoposide, aphidicolin, 5FU, and
purvalanol.
8. The method of claim 1 wherein the tissue or cells are selected
from the group consisting of vulvovaginal tissues and cells and
rectal tissue and cells.
9. The method of claim 1 wherein the tissue or cells are selected
from the group consisting of oral cavity tissue and cells and oral
pharynx tissue and cells.
10. The method of claim 1 wherein an effective amount of a viral
inhibitor selected from the group of G1, S, G2, and M cell cycle
inhibitors is a concentration of at least 1.5 .mu.M.
11. The method of claim 1 wherein an effective amount of a viral
inhibitor selected from the group of G1, S, G2, and M cell cycle
inhibitors is a concentration of at least 0.1 .mu.M.
12. A composition comprising an effective amount of a viral
inhibitor selected from the group of G1, S, G2, and M cell cycle
inhibitors and a pharmaceutically acceptable carrier, wherein the
viral inhibitor is effective for inhibiting papillomavirus
infection.
13. A composition comprising an effective amount of a viral
inhibitor selected from the group of G1, S, G2, and M cell cycle
inhibitors and mixtures thereof and a product designed for
application in the vaginal or rectal areas.
14. A composition of claim 13 wherein the product designed for
application in the vaginal or rectal areas is a spermicide,
lubricant, cream, ointment, solution, powder, impregnated tampon,
rectal or vaginal suppository, pessary, or implant.
15. A composition comprising an effective amount of a viral
inhibitor selected from the group of G1, S, G2, and M cell cycle
inhibitors and mixtures thereof and a product designed for
application by inhalation into the respiratory system.
16. A composition of claim 15 wherein the product designed for
application by inhalation into the respiratory system is a spray,
aerosol, or foam.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority from U.S. provisional
application Ser. No. 61/133,676 filed Jul. 1, 2008 which is
incorporated by reference in its entirety for all purposes.
BACKGROUND OF THE INVENTION
[0003] Human papillomaviruses (HPVs) are DNA viruses associated
with major human cancers. As such there is a strong interest in
developing new means, such as vaccines and microbicides, to prevent
HPV infections. Developing the latter requires a better
understanding of the infectious life cycle of HPVs.
[0004] The HPV infectious life cycle is closely linked to the
differentiation state of the stratified epithelium it infects, with
progeny virus only made in the terminally differentiating
suprabasal compartment. It has long been recognized that HPV must
first establish its infection within the stratum basal; however,
why this is the case has not been understood. In part it may
reflect specificity of expression of entry receptors. However, this
hypothesis could not fully explain the differentiation restriction
of HPV infection, since many cell types can be infected with HPVs
in monolayer cell culture.
[0005] Here, we used chemical biology approaches to reveal that
cell cycle progression through mitosis is critical for HPV
infection. Using infectious HPV16 particles containing the intact
viral genome, G1-synchronized human keratinocytes as hosts, and
early viral gene expression as a readout for infection, we learned
that the recipient cell must enter M phase (mitosis) for HPV
infection to take place. Late M phase inhibitors had no effect on
infection; whereas, G1, S, G2, and early M phase cell cycle
inhibitors efficiently prevented infection. We conclude that, host
cells need to pass through early prophase for successful onset of
transcription of the HPV encapsidated genes. These findings provide
a new understanding for why HPVs initially establish infections in
the basal compartment of stratified epithelia. Only this
compartment of the epithelia contains cells progressing through the
cell cycle, and therefore it is only in these cells that HPVs can
establish their infection. By defining a major condition for cell
susceptibility to HPV infection, these results also have
potentially important implications for HPV control.
[0006] Human Papillomaviruses (HPV), which comprise more than 100
genotypes, are the most prevalent sexually transmitted infection
and are associated with multiple human cancers including all
cervical cancers, many other anogenital cancers, and 25% of head
and neck cancers. HPV has unique life cycle which is closely linked
to epithelial differentiation of skin keratinocytes, with initial
infection occurring only in the undifferentiated proliferating
basal compartment of epithelium and progeny virus production only
in the terminally differentiated suprabasal compartment. So far,
little is known about how host cells restrict the HPV life cycle to
specific stages of skin development. Here, in an effort to screen
HPV small molecule inhibitors, we revealed that cell cycle
progression through mitosis is critical for the establishment of
HPV infection. In addition, our further chemical genetic dissection
of this process showed that early steps of mitosis are required for
HPV infection and early gene expression. Our new finding could
explain why HPV only infects undifferentiated proliferating cells
and provide new leads for the development of preventive and
therapeutic strategies against HPV infection.
[0007] Needed in the art is a better understanding of the
infectious life cycle of HPVs and better prevention methods.
SUMMARY OF INVENTION
[0008] In one embodiment, the present invention is a method of
inhibiting double-stranded DNA virus infection. It relies on the
inventor's observations that G1, S, G2, and M cell cycle inhibitors
inhibit double-stranded DNA viruses.
[0009] In a first aspect, the present invention is a method of
inhibiting double-stranded DNA virus infection. In one embodiment
of the first aspect the method comprises the steps of (a)
identifying an individual in danger of viral infection, and (b)
exposing tissues or cells of the individual that are susceptible to
virus infection to an effective amount of a viral inhibitor,
wherein the inhibitor is selected from the group of G1, S, G2, and
M cell cycle inhibitors.
[0010] In different embodiments of the first aspect, the virus is a
papovavirus, HPV, high risk HPV, low risk HPV, or HPV 16. In other
embodiments of the first aspect, the inhibitor is selected from the
group consisting of etoposide, aphidicolin, 5FU, and purvalanol,
and the tissue or cells are selected from the group consisting of
vulvovaginal tissues and cells, rectal tissues and cells, oral
cavity tissues and cells, and oral pharynx tissue and cells. In
still other embodiments of the first aspect, and effective amount
of a viral inhibitor selected from the group of G1, S, G2, and M
cell cycle inhibitors is a concentration of at least 0.1 .mu.M. In
another embodiment, the concentration is at least 1.5 .mu.M.
[0011] In a second aspect, the present invention is a composition
comprising an effective amount of a viral inhibitor selected from
the group of G1, S, G2, and M cell cycle inhibitors and a
pharmaceutically acceptable carrier, wherein the viral inhibitor is
effective for inhibiting papillomavirus infection.
[0012] In a third aspect, the present invention is a composition
comprising an effective amount of a viral inhibitor selected from
the group of G1, S, G2, and M cell cycle inhibitors and mixtures
thereof and a product designed for application in the vaginal or
rectal areas.
[0013] In one embodiment of the second aspect, the product designed
for application in the vaginal or rectal areas is a spermicide,
lubricant, cream, ointment, solution, powder, impregnated tampon,
rectal or vaginal suppository, pessary, or implant.
[0014] In a third aspect, the present invention is a composition
comprising an effective amount of a viral inhibitor selected from
the group of G1, S, G2, and M cell cycle inhibitors and mixtures
thereof and a product designed for application by inhalation into
the respiratory system.
[0015] In one embodiment of the third aspect, the product designed
for application by inhalation into the respiratory system is a
spray, aerosol, or foam.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1. Selected cell cycle inhibitors abrogate HPV
infection. The reporter RL activity (FIG. 1A) and cell viability
(FIG. 1B) were measured by Renilla Luciferase Assay System
(Promega) and CellTiter-Glo Luminescent cell viability assay
(Promega), respectively. Human keratinocyte HaCaT cells were
inoculated with HPV16 LCR-driven RL containing pseudovirions
hpv16wpA-RL after 4 hours of pre-treatment with 3 .mu.M of each
compound, and reporter expression/cell viability were scored 48
hours later. The expression value is shown as viability to
untreated cells with hpv16wpA-RL.
[0017] FIG. 2. Cell cycle arrest by serum starvation abrogates HPV
infection. (FIG. 2A) After 24 hour incubation in serum-free
Dulbecco's Modified Eagle's Medium (DMEM), HaCaT cells were
infected with HPV pseudovirion hpv16wpA-RL and Renilla Luciferase
(RL) activity was measured after 2 days. For serum conversion,
serum-free DMEM was replaced with 10% fetal bovine serum (FBS)/DMEM
before virus inoculation. The expression value is shown as %
infectivity to untreated hpv16wpA-RL inoculation. The columns
represent the mean, the bars represent the standard deviation.
(FIG. 2B) Flow cytometry was performed to confirm cell cycle arrest
by serum starvation. The solid line indicates the HaCaT cells
incubated with serum-free DMEM for 24 hours and the gray shaded
area indicates control HaCaT cells in DMEM with 10% FBS.
[0018] FIG. 3. Cell cycle progression through M phase is critical
for HPV infection. (FIG. 3A) HaCaT cells were synchronized with
aphidicolin (sets 1, 2, 4, and 5) and etoposide (set 3) 24 hours
before hpv16wpA-RL inoculation. Cell cycle progression was released
in set 1 and kept blocked in set 2 and 3 using aphidicolin and
etoposide, respectively. In set 4, cell cycle progressed through S
phase but arrested at G2 phase by switching the compounds in medium
from aphidicolin to etoposide. In set 5, cell cycle was released
for 20 hours to progress through M phase and arrest at G2 phase by
etoposide addition. Experimental protocols are shown as diagrams.
Solid arrows and dotted arrow indicate aphidicolin and etoposide in
cell culture medium, respectively. Histograms show the results of
flow cytometry analysis following propidium iodide staining at 0,
24, and 48 hour time points using a cell cycle analysis program of
FlowJo. (FIG. 3B) The reporter RL activity was measured by Renilla
Luciferase Assay System (Promega).
[0019] FIG. 4. Cell cycle progression through M phase is critical
for HPV infection. Cell cycle release after 24 hours G1 arrest was
extended to 24 and 30 hours before G2 arrest by etoposide.
Expression of HPV16 early RNA transcripts was measured using E7
specific primers. The columns represent the mean, the bars
represent the standard deviation. We could not detect any signal
above the background in reverse transcriptase-negative controls,
indicating that only mRNA expressed from infected virus could be
detected (data not shown).
[0020] FIG. 5. Late M phase arrest does not affect HPV infection.
(FIG. 5A) HaCaT cells were treated with different concentrations of
monastrol, and hpv16wpA-RL infectivity was assessed as above.
Dotted line is for mock-infected cells. (FIG. 5B) Cell cycle arrest
at M phase by monastrol (100 .mu.M) was confirmed using flow
cytometry. Histograms indicate the cell cycle status of DMSO and
monastrol-treated cells, respectively.
[0021] FIG. 6. Early M phase arrest by CDK1 inhibition interferes
with HPV infection. (FIG. 6A) 293T cells were treated with a CDK1
inhibitor, purvalanol A (3, 6, and 12 .mu.M) and infected with
hpvSEAP. SEAP activity was assayed after 48 hours incubation as
described in the Experimental Procedures section in Example I.
p<0.05, significantly different from the dimethyl sulfoxide
(DMSO) control. The columns represent the mean, the bars represent
the standard deviation. (FIG. 6B) Cell cycle arrest at M phase by
purvalanol A (12 .mu.M) is shown. Histograms indicate the cell
cycle status of DMSO and purvalanol A-treated cells,
respectively.
[0022] FIG. 7. Cell cycle arrest does not inhibit influenza virus.
(FIG. 7A) After 24 hour synchronization with aphidicolin and 4 hour
treatment with etoposide or aphidicolin (3 .mu.M each), 293T cells
were infected with hpv16wpA-RL or an influenza virus vector in
which hemagglutinin and neuraminidase open reading frames in viral
RNA were replaced with those of vesicular stomatitis virus
glycoprotein and RL, respectively (Watanabe et al., J. Virol. 77:
10575-10583). (FIG. 7B) After 24 hour synchronization in serum-free
DMEM, HaCaT cells were infected with hpv16wpA-RL and the
RL-expressing influenza virus as in FIG. 7A in the presence or
absence of FBS. RL activity was measured after 48 hours, as
described in the Experimental Procedures section in Example I,
normalized to RL activity in equivalently inoculated cells
maintained in 10% FBS and expressed in the histograms as %
infectivity. The columns represent the mean, the bars represent the
standard deviation.
[0023] FIG. 8. Selected cell cycle inhibitors abrogate HPV
infection. HPV16 early gene (FIG. 8A) and .beta.-actin expression
(B) was measured by quantitative RT-PCR. 293T cells were inoculated
with wild type HPV16 after 4 hours of pre-treatment with 3 .mu.M of
each compound or with 1:100 dilution of neutralizing antibody
(H16.7E). Total RNA was extracted after 48 hours, and expression
levels of HPV16 RNA transcripts were measured by qRT-PCR using E7,
E2, and E5 sequence specific primers (FIG. 11). The expression
value is shown as % infectivity or % expression to untreated cells
infected with HPV16. We could not detect any signal above the
background in reverse transcriptase-negative controls, indicating
that only nRNA expressed from infected virus could be detected
(data not shown). Due to different characteristics of each primer
set, we observed slight differences in the quantity of mRNAs
detected in the same sample with different primer pairs. Those
differences, while not statistically significant, could reflect
differences in the efficiency of each primer pair amplifying
different subsets of viral early mRNAs. Veratrine, and alkaloid
drug, was one of many small molecule compounds for the secondary
screens. The neutralizing antibody (H16.7E, provided by Neil
Christensen at the Pennsylvania State University, Hershey, Pa.) was
used as a positive control for HPV infection inhibition.
[0024] FIG. 9. Cell cycle progression through M phase is critical
for HPV infection. 293T cells were synchronized with aphidicolin
(sets 1, 2, 4, and 5) and etoposide (set 3) 4 hours before wild
type HPV16 inoculation (see FIG. 3). Cell cycle progression was
released in set 1 and kept blocked in sets 2 and 3 using
aphidicolin and etoposide, respectively. In set 4, cell cycle
progressed through S phase, but arrested at G2 phase by switching
the compounds in medium from aphidicolin to etoposide. In set 5,
cell cycle was released for 20 hours to progress through M phase
and arrest at G2 phase by etoposide addition. Wild type HPV16 gene
expression was measured from total RNA extracts as indicated in
FIG. 8 and in the Experimental Procedures section in Example I.
[0025] FIG. 10. Dose-dependent inhibition response of HPV infection
by cell cycle inhibitors. Shown are results from infection
experiments in human keratinocyte HaCaT cells monitoring the
efficiency of infection by HPV16 LCR-driven Renilla luciferase
containing pseudovirion hpv16wpA-RL in the absence [0 uM, vehicle
only] or increasing concentrations [0.02 to 24 uM for etoposide (A)
and aphidicolin (B); 0.1 to 200 uM for 5-FU (C); and 0.2 to 100 uM
for monastrol(D)]. The Y axis is the infectivity by luminescence as
a function of drug concentration (X axis). All data points
represent the average of values from triplicate samples.
[0026] FIG. 11. Quantitative PCR primers of HPV16 early genes.
(FIG. 11A) Oligonucleotide primers were designed using the Primer3
primer design program. (FIG. 11B) The HPV genomic position of each
primer is indicated by an arrow.
DETAILED DESCRIPTION OF THE INVENTION
[0027] A. In General
[0028] Anogenital HPVs are the most common sexually transmitted
pathogens, and the chief cause of anogenital cancers including
cervical, vaginal, vulvar, penile, and anal cancers. Recently
developed vaccines may provide some level of protection against a
subset of these viruses, however, their use is still limited due to
cost, social acceptance, and access to health care providers. In
one embodiment, the present invention is a method of inhibiting DNA
virus infection comprising the steps of identifying an individual
in danger of viral infection and exposing tissues or cells of the
individual that are susceptible to papillomavirus virus infection
to an effective amount of a viral inhibitor, wherein the inhibitor
is selected from the group of G1, S, G2 and M cell cycle
inhibitors.
[0029] By "effective amount" we mean an amount effective to inhibit
HPV infection preferably 95%, more preferably 99%, and most
preferably 100%.
[0030] In a preferred embodiment, the inhibitor of the present
invention is selected from the group consisting of etoposide,
aphidicolin, 5FU and purvalanol.
[0031] B. Inhibited Viruses
[0032] In one broad embodiment, the present invention is a method
and composition for inhibition of papovaviruses, such as human
papillomaviruses (HPVs). In one version of the present invention,
the method would be used to inhibit all HPVs.
[0033] In another embodiment, the invention would be used to
inhibit high risk HPVs, such as HPV16, 18, 31, 33, 35, 39, 45, 51,
52, 53, 56, 58, 59, 66, 68, 70, 73, 82, and 83. In a specific
embodiment, the invention is used to inhibit HPV 16. In another
embodiment, the invention could be used to inhibit low oncogenic
risk HPVs such as HPV6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, and
89. In still another embodiment, the invention is used to inhibit
combinations of HPV genotypes.
[0034] In another embodiment, the present invention is used to
inhibit double strand DNA viruses. By "double strand DNA virus" we
mean all other papillomaviruses and other viruses that require cell
cycle progression for infection and/or replication such as
polyomaviruses, herpesviruses, and adenoviruses.
[0035] In yet another embodiment, the present invention is used to
inhibit retroviruses. By "retrovirus" we mean both simple and
complex retroviruses.
[0036] HPVs are DNA viruses are associated with major human
cancers. We envision that other oncogenic viruses, particularly
oncogenic DNA viruses and oncogenic retro viruses, would be
excellent candidates for the present invention.
[0037] C. Cell Cycle Progression
[0038] The HPV life cycle is closely linked to differentiation of
the stratified epithelium the virus infects. HPV only infects
proliferating basal cells, and progeny virions only produce as
daughter cells terminally differentiate. However, the crucial early
steps of HPV infection from cell binding through nuclear delivery
of viral DNA for early gene expression remain poorly understood,
and the basis of HPV infection specificity for dividing cells is
unknown.
[0039] In the Examples below we used chemical biology approaches to
reveal that cell cycle progression through mitosis is critical for
HPV infection. Further analysis showed that, after HPV attachment
and entry, recipient cells must pass through early prophase, but
not late prophase and metaphase, to allow transcription of
HPV-encapsidated genes in the nucleus. These requirements parallel
those of simple retroviruses that enter via nuclear envelope
breakdown (NEBD). The results were experimentally distinct from
those of influenza virus, which uses active transport through
nuclear pores to deliver its genome to intact nuclei.
[0040] Therefore, the inventors propose the use of G1/S and G2/M
inhibitors for viral inhibition.
[0041] D. Preferred Inhibitors
[0042] The example below discloses the elucidation of criteria for
preferred antiviral compounds. Example I demonstrates that cell
cycle progression through mitosis is critical for HPV infection.
Our analysis showed that recipient cells must pass through early
prophase but not late prophase and metaphase to allow transcription
of HPV encapsidated genes in the nucleus.
[0043] Thus, we have learned that inhibitors of the cell cycle at
G1/H and G2 stage are effective antivirals. These small molecules
target multiple cyclin-dependent kinases such as CDK1 and CDK2. The
examples below show that aphidicolin and etopside significantly and
consistently inhibit HPV 16. Therefore, in one embodiment of the
present invention the cell cycle inhibitors are aphidicolin or
etopside. In another embodiment, the cell cycle inhibitor is an
G1/S and/or G2/M inhibitor. Other examples of G1/S and G2/M
inhibitors are as follows: [0044] G1/S inhibitors: Aphidicolin,
5-Flourouracil, Daidzein, Netropsin, XK469, Sobuzoxane,
Amethopterin, PD 0183812, Fascaplysin, L-Mimosine- [0045] G2/M
inhibitors: Etoposide, Purvalanol, Apigenin, Indirubin-3'-oxime,
Staurosporine, Scytonemin, Camptothecin. BI 2536, and CGP-74514A.
[0046] G1/S and G2/M inhibitors: Olomoucine, Roscovitine, NU2058,
NU6027, Flavopiridol, and Butyrolactone.
[0047] E. Tissues And Cells
[0048] In one embodiment, the present invention is a method of
inhibiting viruses and blocking the establishment of viral
infection. Preferably, the virus is either HPV or one of the
viruses discussed above. In a preferred embodiment of the present
invention, one would apply the inhibitor to a skin surface area
susceptible to viral infection.
[0049] In one embodiment of the present invention, one would
administer an inhibitor selected from the group consisting of G1/S
and GM/2 inhibitors to a susceptible tissue or cell. By
"susceptible tissue or cell", we mean a tissue or cell that is
capable of infection by a double strand DNA virus, preferably a
papillomavirus, more preferably HPV. So far, the only confirmed way
of HPV transmission is direct and indirect skin contact, including
sexual activities. However, as HPV also infects mouth and throat
tissue and recent studies showed some lung cancers are associated
with HPV infection, HPV transmission might be much more complicated
than we know now. Additional preferred tissues would be those of
the oral cavity and oral pharynx.
[0050] F. Preferred Methods
[0051] In one embodiment, if one wished to prevent HPV infection,
one would add the inhibitor to a spermicide, lubricant, or other
product designed for application in the vaginal area. In another
preferred embodiment, the pharmaceutical composition may be
selected from the group consisting of rectal or vaginal
suppositories, ointments, solutions, powers, and impregnated
tampons. In another embodiment of the present invention, the
pharmaceutical composition is administered as a spray, aerosol, or
foam.
[0052] Vaginal or vulvovaginal delivery of a medication may be by a
device, such as disclosed in U.S. Ser. No. 11/763,085 and
11/454,604. A "vulvovaginal surface" herein denotes any external or
internal surface of the female genitalia, including mucosal
surfaces in the vaginal cavity and nonmucosal surfaces of the vulva
and immediately surrounding areas of skin. In some embodiments, the
composition is more specifically adapted for application to a
vaginal mucosal surface, and an external phase of the composition
is bioadhesive to such a surface.
[0053] A composition used in methods of the invention can be in any
suitable form that is adapted for vulvovaginal administration. For
intravaginal administration, suitable forms include a vaginal
cream, tablet, suppository, pessary or implant, but in particular
embodiments, the composition is in the form of a vaginal cream.
[0054] The composition can be administered topically to external
surfaces of skin surface, preferably the vulva and/or to
surrounding areas of skin. In addition or alternatively, the
composition can be administered intravaginally. In one embodiment,
the composition is a vaginal cream, i.e., a semi-solid formulation
adapted for administration to vaginal mucosal surfaces.
[0055] A vaginal cream for use according to methods of the
invention can be administered to contact a mucosal surface in the
vaginal cavity by means, for example, of an applicator that is
optionally pre-filled with a single unit dosage amount of the
cream. With the patient optionally in a supine position, the tip of
the applicator can be gently inserted high in the vagina, for
example in the posterior vaginal formix, and the cream can be
released through the tip by pushing on a plunger of the
applicator.
[0056] In some embodiments anal or rectal delivery of the inhibitor
would be preferred. Suitable formulations for rectal administration
include, for example, suppositories, which consist of the packaged
inhibitor with a suppository base. Suitable suppository bases
include natural or synthetic triglycerides or paraffin
hydrocarbons. In addition, it is also possible to use gelatin
rectal capsules which consist of a combination of the packaged
inhibitor with a base, including, for example, liquid
triglycerides, polyethylene glycols, and paraffin hydrocarbons.
Another option is the use of penile lubricants.
[0057] If one is concerned about protection about other categories
of viruses, one would apply the inhibitor as follows: For
respiratory viruses (e.g. adenoviruses) application may be via
inhaled mist. For enteric viruses, delivery would be via oral
route. For other viruses, such as arthropod borne viruses, systemic
delivery might be appropriate.
[0058] F. Dose And Concentration
[0059] In one embodiment of the present invention, determining a
proposed human dose would first involve testing in the mouse model
doses in existing or modified vehicle formulations currently used
in vaginal/penile lubricants and contraceptive jellies/creams. If
necessary, one would make tolerated modifications to the drugs to
improve their solubility in these vehicle formulations.
[0060] In another embodiment of the present invention, the proposed
human dose is a compound concentration of at least 1.5 .mu.M. In a
preferred embodiment, the concentration is at least 0.1 .mu.M.
Example I
Establishment of Human Papillomavirus Infection Requires Cell Cycle
Progression
[0061] The tight link between keratinocyte differentiation and the
HPV life cycle has made large scale production of mature infectious
HPV particles difficult, greatly restricting studies of the
mechanisms of natural HPV infection (Dollard S. C. et al. (1992)
OFF. Genes Dev. 6: 1131-1142; Meyers C. et al., (1992) Science 257:
971-973). Recently developed transfection methods that generate
large yields of virus particles and efficient encapsidation of
target plasmids as large as the full length .about.8 kb HPV genome
have overcome this limitation (Buck C. B. et al., (2004) J. Virol
78: 751-757; Pyeon D, et al., (2005) Proc. Nat'l Acad. Sci. USA
102: 9311-9316. Epub 2005 June 9315).
[0062] This technique provides a genetically modifiable, high yield
source of infectious HPV and HPV pseudoviruses expressing reporter
genes for studies of different early steps of HPV infection
including virion binding, endocytosis, uncoating of the virion
capsid, release from the endosome, trafficking to the nucleus,
delivery of viral DNA to the nucleus, and transcription of
encapsidated genes.
[0063] To define the pathways and mechanisms involved in these
early steps of HPV infection, we tested approximately 5,000
bioactive compounds with known mechanisms of action for effects on
the entry of HPV capsids containing reporter genes or the full HPV
genome, and identified a subset of cell cycle inhibitors that
completely blocked wild type HPV infection. Our further studies
showed that cell cycle progression through early stages of mitosis
is critical for successful HPV infection. These findings reveal new
insights into the mechanism by which HPV infects cells and provide
one reason why HPV infects only undifferentiated, proliferating
cells. These results also provide new leads for developing
preventative and therapeutic strategies against HPV infection.
[0064] Results
[0065] Cell cycle inhibitors block HPV infection. To identify
mechanistic pathways and informative modulators of HPV infection,
we tested nearly 5,000 compounds in known bioactive molecule
libraries Prestwick (Prestwick Chemicals) and LOPAC (Library of
Pharmacologically Active Compounds, Sigma), with HPV pseudovirions
containing an SV40 promoter-driven secreted alkaline phosphatase
(SEAP) reporter gene (hpvSEAP). 293T cells were infected with wild
type HPV16 after 4 hours of pre-treatment with 3 .mu.M of each
compound or with 1:100 dilution of neutralizing antibody (H16.7E).
Total RNA was extracted after 48 hours, and expression levels of
HPV16 E1 E4 RNA transcripts were measured by qRT-PCR using E7, E2,
and E5 sequence specific primers.
[0066] High activity effectors identified in this assay were
retested in a second infectivity assay using HPV pseudovirions
containing Renilla luciferase driven by the natural HPV16 promoter
(hpv16wpA-RL) to exclude false positive compounds that may directly
affect the SV40 promoter or alkaline phosphatase (data not shown).
Secondary screens performed in HaCaT cells, an immortalized line of
human keratinocytes, which are natural host cells for HPV
infection, confirmed the validity of the hits, as indicated
below.
[0067] Among the confirmed hits identified in the primary screen
were cell cycle inhibitors etoposide (Pedrali-Noy G. et al., (1980)
Nucleic Acids Res. 8:377-387) and aphidicolin (Lock R. B. and Ross
W. E. (1990) Cancer Res. 50:3761-3766) showed the most significant
and consistent inhibition on HPV infection in 293 cells both using
HPV16 pseudoviruses expressing SEAP or Renilla luciferase reporter
genes, or using intact infectious HPV16 virus in which we scored
for early gene expression (FIG. 1). The levels of inhibition
achieved with these drugs was greater than that achieved with
neutralizing antibody to HPV16 (FIG. 1A), and were specific to
virally expressed gene as the drugs had no effect on cellular
.beta.-actin expression (FIG. 1B).
[0068] Our further testing with wild type HPV16 virion infection of
293T cells showed that aphidicolin and etoposide blocked HPV16 gene
expression even more efficiently than a neutralizing antibody (FIG.
8A), while cellular 13-actin expression was not affected (FIG. 8B).
We examined cell cycle status using flow cytometry and found that 3
.mu.M aphidicolin or etoposide, which block HPV infection, arrested
cell cycle progression at G1/S and G2 phases, respectively (data
not shown), in keeping with their known mechanisms of action
(Pedrali-Noy et al., Nucleic Acids Res. (1980) 8: 377-387; Lock et
al., Cancer Res. (1990) 50: 3761-3766).
[0069] In the more physiologically relevant HaCaT cells, these two
drugs along with another cell cycle inhibitor 5-fluorouracil (5-FU)
(Lewin F. et al., (1987) Acta Oncol. 26:125-131), all were very
robust in identifying infectivity (FIG. 1A) at concentrations that
did not have any effect on cell viability (FIG. 1B). These results
demonstrate that the original screen, which was done in 293T cells,
was successful in identifying drugs that can inhibit HPV infection
in a more relevant cell type. More importantly our results
suggested that the cell cycle is critical for HPV infection.
[0070] Next, we arrested cell cycle using a non-chemical method
with serum starvation. HaCaT cells incubated in serum free medium
showed complete cell cycle arrest and significant inhibition of HPV
infection (FIG. 2A) in efficient cell cycle arrest at G1 phase
(FIG. 2B). However, when cell cycle was released upon HPV
inoculation, HPV infection was not inhibited, but enhanced about
two-fold. These results consistently indicate that cell cycle
progression is necessary for HPV early infection and viral gene
expression.
[0071] Cell cycle progression through mitosis is critical for HPV
infection. To define which cell cycle step(s) is critical for early
HPV infection, we synchronized HaCaT cells with aphidicolin or
etoposide and then tightly controlled cell cycle progression. In
the first set of the experiments (indicated as No inhibition; FIGS.
3A and 3B), cells were synchronized in G1 phase with a 24 hour
aphidicolin treatment, inoculated with hpv16wpA-RL virions, and
then 4 hours later, released by removing aphidicolin from the
culture medium. After 48 hour incubation, the infected HaCaT cells
expressed high levels of RL. The reporter gene expression levels
under these "no inhibition" positive control conditions were set to
100% infection and used to normalize the early gene expression
levels from the other experimental conditions (FIG. 9).
[0072] In the second (G1 arrest; FIGS. 3A and 3B) and third (G2
arrest; FIGS. 3A and 3B) conditions used, cells were treated with
either aphidicolin or etoposide for 24 hours, after which
hpv16wpA-RL virions were added. The cell cycle was blocked at G1/S
and G2 by aphidicolin and etoposide, respectively, and HPV
infection was significantly and consistently inhibited under both
conditions (FIGS. 3A and 3B). These conditions were the same as
used in FIG. 1, and the flow cytometric analyses shown in FIG. 3
confirmed effective cell cycle block by aphidicolin and etoposide
at the concentrations used in FIG. 1.
[0073] In the fourth condition (S phase progress; FIGS. 3A and 3B),
we synchronized the cells in G1/S phase by 24 hour aphidicolin
treatment, then added hpv16wpA-RL virions and, four hours later,
replaced aphidicolin with etoposide. When aphidicolin was replaced
by etoposide, cell cycle progressed from G1 arrest through S phase
to G2 arrest, allowing us to test whether S phase progression
supports HPV early infection. However, this experiment showed no
difference in HPV gene expression with G1/S or G2 arrest by
aphidicolin and etoposide (FIGS. 3A and 3B).
[0074] In the fifth condition (S & M phase progress; FIGS. 3A
and 3B), after 24 hour synchronization and subsequent hpv16wpA-RL
virion addition, the cell cycle was released for 20 hours, followed
by cell cycle arrest in G2 phase with etoposide, allowing the cells
to progress through one complete round of the cell cycle, including
M phase. Interestingly, one round of M phase progression was
sufficient for HPV16 infection and gene expression (FIGS. 3A and
3B).
[0075] Interestingly, one round of M phase progression was
sufficient for HPV infection and gene expression (FIGS. 3A and 3B).
Our further testing with wild type HPV16 virions confirmed that one
round of cell cycle progression through M phase is sufficient for
HPV early gene expression (FIG. 2). These results imply that cell
cycle progression through mitosis is critical for early steps of
HPV infection. Consistent with this premise, HPV gene expression
levels were further enhanced when the cell cycle was released for
progressively longer periods before G2 arrest, eventually reaching
levels well above the control "no arrest" conditions (FIG. 4). This
suggests that G2 arrest might provide better host cell environment
for viral gene expression once the virus enters nucleus.
[0076] Early prophase, but not late prophase or metaphase, is
critical for HPV early infection. To define further the states of
mitosis and associated cellular functions that are not required to
support HPV early infection, we tested monastrol (Mayer et al.,
Science (1999) 286:971-974), which inhibits mitotic spindle
formation during late prophase and metaphase in early to mid
mitosis. Interestingly, monastrol, which induced cell cycle arrest
in M as expected (FIG. 5B) showed no inhibitory effect on HPV
infection in HaCaT cells (FIG. 5A). At high concentrations of
monastrol, there was a slight, but reproducible increase in HPV
infectivity. Thus, HPV infection can efficiently arise in cells
inhibited in late prophase/metaphase. These results, taken together
with those in FIG. 3 narrowed down the critical stage of the cell
cycle for HPV infection to likely be early prophase.
[0077] Cellular events in early prophase include nuclear envelope
(NE) breakdown and changes in chromatin structure. Phosphorylation
of NE components by CDK1 is critical for these events in early
prophase (Collas, J. Cell Sci. (1999) 112:977-987; Peter et al.,
(1990) Cell 61:591-602; Santamaria et al., Nature (2007)
448:811-815). Thus, to examine whether entry into early prophase is
critical for HPV infection, we tested hpvSEAP infection in the 293T
cells treated with the known CDK1 inhibitor, purvalanol A (Gray et
al., (1998) Science 281: 533-538). Purvalanol A inhibited HPV
infection dose-specifically, with 12 .mu.M both significantly
arresting the cell cycle at G2/M phase and inhibiting HPV infection
about 5-fold (FIGS. 6A and 6B). These results imply that cellular
event(s) exclusively happening in early prophase are critical for
HPV infection to deliver virions into the nucleus and early gene
expression.
[0078] Influenza virus genome delivery via nuclear pores is
unaffected by cell cycle inhibitors that block HPV infection. HPV
minor capsid protein L2 has nuclear localization signals, and,
consequently, it has been hypothesized that L2 directs nuclear
pore-mediated import of HPV DNA during infection (Fay et al., J.
Virol (2004) 78:13447-13454). One virus that is well established to
import its genome into intact nuclei via nuclear pores is influenza
virus (Kemler et al., Virology (1994) 202:1028-1033; Neumann et
al., J. Virol (1997) 71:9690-9700). To test whether HPV and
influenza virus might share similar nuclear entry mechanisms, we
treated 293T cells in parallel with either recombinant influenza
virus or Renilla luciferase-expressing HPV pseudovirions in the
presence and absence of etoposide and aphidicolin. While etoposide
and aphidicolin efficiently blocked HPV infection, as before, they
did not significantly affect influenza infection (FIG. 7A), showing
that their effects on HPV could not be explained by effects on
nuclear pores. We also observed in HaCaT cells the same difference
in cell cycle dependency when those cells were arrested by serum
and inoculated with the same Renilla luciferase-expressing HPV
pseudovirions or recombinant influenza virus. Under these
conditions (FIG. 7B), HPV infection was inhibited over 50 fold
while influenza virus was only reduced approximately 2-fold, likely
due to the general suppression of cell metabolism upon serum
starvation.
[0079] Discussion
[0080] HPVs are clinically important pathogens due to their strong
association with multiple human malignancies and because they are
the most common sexually transmitted pathogens. Some oncogenic
mechanisms of HPV are well known, especially pathways involved with
E6 and E7 viral oncoproteins (Hebner et al., Rev. Med. Virol.
(2006) 16: 83-97). However, many other basic mechanisms of the HPV
life cycle are largely unknown including how HPV selectively
effects only undifferentiated basal cells. Here, we define one of
the bases for this restriction by showing that cell cycle
progression into mitosis and, in particular, events in early
prophase are critical for HPV early infection.
[0081] Viruses employ varied strategies to deliver their genetic
material to the nucleus for replication and viral gene expression
(Hebner et al., Rev. Med. Virol. (2006) 16: 83-97). While
adenoviruses dissociate their capsids in the cytoplasm and import
naked genomic DNA into the nucleus, herpesvirus virions release
their genome into the nucleus without any extensive capsid
disassembly, and polyomaviruses and parvoviruses transport the
entire capsid into the nucleus. The nuclear import strategies of
papillomaviruses have been largely unexplored, mainly because the
means for producing high yields of infectious virus have only
recently been attained (Buck et al., J. Virol. (2004) 78:751-757;
Pyeon et al., Proc. Nat'l Acad. Sci. USA (2005) 102:9311-9316. Epub
2005 June 9315).
[0082] In the present study, using infectious wild type HPV16
virions, we found that events in the early prophase segment of
mitosis are critical for establishing HPV infection, as assayed by
introduction and expression of HPV-encapsidated DNA in the nucleus.
As discussed below, these events could include nuclear envelope
breakdown, cytoskeleton restructuring, and subnuclear structure
changes as well as the specific expression of one or more genes or
gene combinations in early mitosis.
[0083] One critical event in early phases of mitosis is nuclear
envelope (NE) breakdown. This is triggered by a signal cascade that
involves polo I like kinase 1 (plk1) and cyclin B1-associated CDK1
(Collas, J. Cell Sci. (1999) 112:977-987; Peter et al., Cell (1990)
61:591-602; Burkard et al., Proc. Nat'l Acad. Sci. USA (2007)
104:4383-4388. Epub 2007 March 4386). Using a chemical inhibitor of
CDK1, purvalanol A (Gray et al., Science (1998) 281: 533-538), we
found that CDK1 activity is required for efficient infection by
HPVs (FIG. 6A). Therefore, our data are consistent with the
hypothesis that NE breakdown is necessary for the HPV encapsidated
DNAs to enter the nucleus. This is similar to the strategy thought
to be employed by simple retroviruses to allow nuclear entry and
integration of their proviral genomes (Bieniasz et al., Cell cycle
dependence of foamy retrovirus infection. J. Virol. 69: 7295-7299
(1995); Lewis et al., J. Virol. (1994) 68: 510-516).
[0084] The process that initiates nuclear envelope breakdown is not
fully understood. While the phosphorylation of NE components by
CDK1 correlates with NE breakdown, and inhibition of cdk1 prevents
NE breakdown [Collas, J. Cell Sci. (1999) 112:977-987; Peter et
al., Cell (1990) 61:591-602), some have argued that NE breakdown is
initiated by mechanical tension induced by spindle microtubules,
leading to holes in the NE (Margalit et al., J. Cell Biochem.
(2005) 95:454-465; Salina et al., Cell (2002) 108:97-107).
Consistent with this hypothesis, the microtubule minus end motor,
dynein, translocates to the outer face of the NE just before NE
breakdown. This model for NE breakdown by mechanical tension has an
interesting connection to HPV infection.
[0085] L2 binds to dynein and this is thought to allow L2:HPV DNA
complexes to be translocated along microtubules towards the nucleus
(Florin, J. Virol. (2006) 80:6691-6696). Thus the dynein-mediated
trafficking of HPV encapsidated DNA cargo may not just contribute
to movement of the encapsidated DNA to the intracytoplasmic
destination of dynein, but microtubule organizing center, but might
also contribute to nuclear localization if the above described
model for NE breakdown is correct, as originally proposed
[0086] Another notable cellular event in early mitosis is the
reorganization of cytoplasmic microtubules to support mitotic
spindle formation, chromosome segregation, and cell division
(Hughes et al., PLoS Biol. (2008) 6:e98; Luders et al., Nat. Rev.
Mol. Cell Biol. (2007) 8:161-167). Like many other viruses, HPV
also utilizes microtubule structure to be delivered from cell
surface to the nucleus (Florin, J. Virol. (2006) 80:6691-6696),
although many details of this process remain to be understood. Our
results are consistent with the possibility that effective HPV
entry and/or HPV DNA delivery require cell cycle-associated
reorganization of microtubules, such as the interaction of
microtubules with host chromosomes in early mitosis.
[0087] In addition, restructuring of PML oncogenic bodies (PODs)
and chromatin in early mitosis could be necessary for establishing
HPV infection in the nucleus. PODs, also know as ND10, are multiple
subnuclear bodies implicated in multiple cellular functions
including transcription, DNA repair, viral defense, stress, cell
cycle regulation, proteolysis and apoptosis (Borden, Mol. Cell
Biol. (2002) 22:5259-5269; Ching et al., J. Cell Sci. (2005)
118:847-854). PODs are also the final destination of HPV DNA during
the initial steps in infection and sites at or near which HPV DNA
replication and transcription occur (Van Tine B. A., et al., J.
Virol. (2004) 78:11172-11186; Day et al., Proc. Nat'l Acad. Sci.
USA (2004) 101:14252-14257 Epub 12004 September 14221). PODs are
dynamically restructured during S and M phases, responding to
changes in chromatin organization (Bernardi et al., Nat. Rev. Mol.
Cell Biol. (2007) 8:1006-1016). Thus, structural changes of PODs in
early mitosis might be critical for HPV DNA localization to its POD
destinations during the establishment of HPV infection. Other host
mechanisms associated with chromatin structure in early mitosis
might also contribute to structural changes of the incoming HPV DNA
and thereby facilitate viral gene expression and replication inside
the nucleus. Little is currently known about HPV chromatin
structure and its contribution to viral gene expression.
[0088] Many genes such as polo-like kinases and aurora kinases are
exclusively expressed only in early mitosis to certify completion
of DNA replication, overcome mitotic checkpoint, and initiate
mitosis (Ferrari, Cell Mol. Life Sci. (2006) 63:781-795). Beyond
the potential mechanisms of cell cycle dependence suggested above,
host genes or gene combinations specifically expressed only in
early mitosis might have essential roles for HPV entry steps and/or
HPV early gene expression.
[0089] HPV is thought to be able to infect the proliferating basal
layer of the stratified epithelium only when wounds allow HPV to
penetrate the physical barrier of upper skin layers. Wounding can
also provide HPV particles access to laminin 5 or heparin sulfate
moieties, both components of the extra cellular matrix (ECM)
component of the epithelial basement membrane for which HPV
particles have high affinity, thereby localizing the virus
particles to basal surface epithelial cells it needs to infect
(Culp T. D. et al., Virology (2006) 347:147-159 Epub 2005 December
2027; Culp T. D., et al., J. Virol. (2006) 80:8940-8950).
[0090] Laminin 5 is also the ECM partner of integrin
.alpha.6/.beta.4, which has been reported by some investigators to
be an entry receptor for HPVs ((Culp T. D. et al, Virology (2006)
347:147-159 Epub 2005 December 2027; Culp T. D., et al., J. Virol.
(2006) 80:8940-8950; Evander M. et al., J. Virol. (1997)
71:2449-2456). Our results suggest that wound healing might
increase the efficiency with which the HPV DNA becomes established
as a nuclear plasmid in the basal cells, because the basal cells
are then in a hyperproliferative state (Werner S, et al., Science
(1994) 266:819-822). HPV may also provide a mitogenic signal upon
binding to cell surface receptors (Fothergill et al., Virology
(2006) 352:319-328. Epub 2006 June 2015; Payne et al., J. Virol.
(2001) 75:4150-4157), though if so, that signal is insufficient to
overcome serum starvation-induced cell cycle arrest in HaCaT cells
(FIGS. 2A and 2B). Regardless of why the cell is cycling, our data
indicate that its movement through mitosis is a critical step in
establishing HPV infection.
[0091] To further confirm physiological relevance regarding the
cell cycle dependence of HPV infection, it would be necessary to
examine these results in primary keratinocytes and in vivo models
such as mouse and non-human primate. In addition, real-time
particle imaging studies could be employed to identify what
specific steps in infection are blocked upon cell cycle arrest;
however, the ability to interpret accurately such imaging studies
rests on the ability to distinguish particles leading to infectious
events from the vast majority of particles that give rise to
abortive or non-infectious events. To date no imaging method exists
able to make such a distinction at the individual particle
level.
[0092] The recent development of HPV vaccines offers an extremely
important avenue for control of HPV infections and their associated
cancers (Schiller J. J. Nat. Rev. Microbiol. (2004) 2:343-347).
Nevertheless, due to limitations of recently approved HPV vaccines,
there is an urgent need to identify other approaches to prevent HPV
infections. One potentially valuable approach to prevent STDs
including genital HPV infections could be ready access to effective
microbicides (Howett et al., Curr. Pharm. Des. (2005)
11:3731-3746). From this perspective, our findings provide a new
target mechanism for preventing initial HPV infection and blocking
further spread. In addition, our chemical biology screening
approach has successfully identified compounds that inhibit HPV
infection, and the associated infection assay appears promising for
large scale screening of compound libraries for development of
further HPV preventives and therapeutics.
[0093] Experimental Procedures
[0094] Plasmids. pEF399, containing the complete W12E HPV16 genome
(Flores et al., Virology (1999) 262:344-354), and all other
plasmids used for virus packaging were previously described (Pyeon
et al., Proc. Nat'l Acad. Sci. USA (2005) 102:9311-9316. Epub 2005
June 9315). pSEAP (pSEAP-control) for expression of secreted
alkaline phosphatase (SEAP) was purchased from Clontech.
pHPV16wpA-RL was cloned with HPV16 long control region (LCR,
nucleotide position 7155-861), into pRL-null (Promega) to prepare a
Renilla luciferase reporter system driven by native HPV16
promoter.
[0095] Cell lines. Human embryonic kidney cell line 293T from ATCC,
and its enhanced SV40 T antigen-expressing daughter cell line 293TT
(Buck et al., J. Virol. (2004) 78:751-757) from John Schiller, were
maintained in Dulbecco's modified eagle's medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) (Invitrogen).
Immortalized human keratinocyte cell line HaCaT (Boukamp et al., J.
Cell Biol. (1988) 106:761-771) was maintained in F-media
(Invitrogen, 3 parts F-12 and 1 part DMEM), supplemented with 10%
FBS.
[0096] Production of virus particles. HPV virions and pseudovirions
were prepared as described previously Pyeon et al., Proc. Nat'l
Acad. Sci. USA (2005) 102:9311-9316. Epub 2005 June 9315]. Briefly,
we cotransfected 293TT (provided by John Schiller) cells with HPV16
capsid protein expression plasmid as well as one of the target DNAs
for encapsidation. After 48 hours at 37.degree. C., cells were
harvested and virions were purified using Optiprep gradient
centrifugation. Influenza virus containing RL (Watanabe et al., J.
Virol. (2003) 77:10575-10583) was provided by Yoshihiro
Kawaoka.
[0097] Small molecule compounds and libraries. Known Bioactive
Library (KBA01) consists of 3 commercially available collections
totaling 4,160 compounds. KBA01 consists of 880 high purity
compounds of known safety and bioavailability in humans of which
over 85% are marketed drugs from Prestwick Chemical. The Prestwick
compounds cover several therapeutic areas including
neuropsychiatry, cardiology, immunology, inflammation, analgesia,
etc.
[0098] Also included in the KBA01 library is 2000 diverse FDA
approved drugs and natural products from Spectrum Chemical
Collection From Microscource Discovery Systems, Inc. and 1280
compounds from the LOPAC of Sigma representing marketed drugs,
failed development candidates and "gold standards" that have
well-characterized activities. These compounds are the results of
lead optimization efforts and have been rationally designed by
structure-activity relationship studies. The NC103 library consists
of 235 natural products that were selected from the NCI open
repository on the basis of structural diversity and availability of
compound. Etoposide, Aphidicolin, Monastrol, 5-Fluorouracil, and
CGP-74514A were purchased from Sigma.
[0099] High throughput screening. 293TT cells in 384-well plates
were treated with 3.3 .mu.M of library compounds for 4 hours and
infected with hpvSEAP at 50 vge (viral genome equivalent)/cell for
48 hours. Culture supernatant 2.5 .mu.l was used for virus
infectivity assay using a Phospha-Light, a chemiluminescent
alkaline phosphatase assay, (Applied Biosystems) and cell lysate
was used for cell viability assay using CellTiter-Glo Luminescent
cell viability assay (Promega). Biomek FX (Beckman Coulter) was
used for automated liquid handling and a Victor 3-V plate reader
(Perkin Elmer) was used for measuring luminescence. Screening was
performed in the Small Molecule Screening Facility at the
University of Wisconsin Comprehensive Cancer Center.
[0100] Infectivity assay. The activity of
pSEAP-control-encapsidated pseudovirions (hpvSEAP) was measured
using a Phospha-Light a chemiluminescent alkaline phosphatase assay
(Applied Biosystems) and the activity of pHPV16wpA-RL-encapsidating
pseudovirions (hpv16wpA-RL) was assayed using Renilla Luciferase
Assay System (Promega). Infectivity of wild type HPV16 virions was
examined by quantitative reverse transcriptase PCR (qRT-PCR) by
amplifying viral mRNA signals from HPV16-treated 293TT cells. 293TT
cells were infected with 50-100 vge/cell of packaged virus and
incubated for 48 hours at 37.degree. C.
[0101] Total RNA was isolated using the RNeasy total RNA
purification kit (Qiagen), treated with RQ DNasel (Promega) to
remove possible DNA contaminants, purified again on RNeasy columns
to remove DNasel, and quantified by spectrophotometer. cDNA was
synthesized from 20 .mu.g of total RNA with oligo (dT) using a
SuperScript cDNA synthesis kit (Invitrogen), and qPCR was performed
with QuantiTect SYBR Green PCR Kit (Qiagen). Oligonucleotide
primers (FIG. 3) were designed using the Primer3 primer design
program (Rozen et al., In: Krawetz S, Misener S, editors.
Bioinformatics Methods and Protocols: Methods in Molecular Biology.
Totowa, N.J.: Humana Press. pp. 365-386 (2000)), synthesized by MWG
and used at 0.5 .mu.M for PCR amplification for 40 cycles of 30
second denaturation at 94.degree. C., 30 seconds annealing at
55.degree. C., and 30 seconds polymerization at 72.degree. C.
Obtained values were normalized with the levels of
.beta.-actin.
[0102] Flow cytometry. The cell cycle status of treated and
untreated cells was analyzed using a propidium iodide (PI)
incorporation method. Briefly, HaCaT cells were harvested,
homogenized, fixed with cold 70% ethanol for 30 minutes at
-20.degree. C., and incubated with PI staining solution (1 mg/ml
RNase A, 33 .mu.g/ml PI, 0.2% NP-40 in PBS) for 30 minutes at room
temperature. Stained cells were filtered through 35 .mu.m-pore
nylon mesh cell filtering caps (Falcon) and analyzed by flow
cytometry using Becton Dickinson FACSCalibur (488 nm laser
excitation).
Sequence CWU 1
1
6122DNAArtificial SequenceSynthetic oligonucleotide 1aaatgacagc
tcagaggagg ag 22222DNAArtificial SequenceSynthetic oligonucleotide
2gagtcacact tgcaacaaaa gg 22322DNAArtificial SequenceSynthetic
oligonucleotide 3actatccagc gaccaagatc ag 22422DNAArtificial
SequenceSynthetic oligonucleotide 4tgttaaatgc agtgaggatt gg
22522DNAArtificial SequenceSynthetic oligonucleotide 5tttgtgtgct
tttgtgtgtc tg 22622DNAArtificial SequenceSynthetic oligonucleotide
6agaggctgct gttatccaca at 22
* * * * *